Κ-carrageenan Concentration Research Articles

Aggregation of casein micelles and κ-carrageenan in reconstituted skim milk

A novel method to form casein/ κ-carrageenan aggregates in skim milk, by mixing solutions of each at 60 °C and cooling to 25 °C while shearing, was investigated. The average particle size of casein/ κ-carrageenan aggregates decreased with increasing shear rate (200, 400 and 800 s −1) but increased with κ-carrageenan concentration (0.025% 0.05% and 0.075%). Oscillatory rheology measurements demonstrated that skim milk containing 0.025% κ-carrageenan behaved as a dilute solution, while skim milk with 0.05% or 0.075% κ-carrageenan formed weak gels, although solutions remained fluid and were at a much lower modulus compared to similar solutions cooled with no shearing. The aggregates were quite stable to shear once formed. The initial mixing temperature of reconstituted skim milk and κ-carrageenan dispersions greatly affected the particle size distribution of the aggregates. Microstructural observations as well as studies in the presence of NaI demonstrated that at particular protein/polysaccharide ratios, casein/ κ-carrageenan aggregates formed in reconstituted skim milk via adsorption of κ-carrageenan with casein micelles.

Physicochemical Characterization of Papain Entrapped in Ionotropically Cross-Linked Kappa-Carrageenan Gel Beads for Stability Improvement Using Doehlert Shell Design

This work examines the influence of various process parameters on papain entrapped in cross-linked κ-carrageenan beads for improvement of its stability. A Doehlert shell design (DSD) was employed to investigate the effect of three process variables, namely κ-carrageenan concentration, KCl concentration, and hardening time, on the entrapment, time required for 50% enzyme release (T50), time required for 90% enzyme release (T90), and particle size. The beads were prepared by dropping the κ-carrageenan containing papain into a magnetically stirred KCl solution. Topographical characterization was carried out by scanning electron microscopy and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zone III and IV. A polymeric matrix was prepared with κ-carrageenan (3.5% w/v) and potassium chloride (0.5 M) using the ionotropic gelation method, with a hardening time of 20 min. Beads characterized by a spherical disc shape with a collapsed center, an absence of aggregates, an entrapment of 82.75%, a T90 value of 55.36 min, and a composite index of 88.55 were produced. The shelf-life of the enzyme-loaded beads was found to increase to 3.63 years compared with 1.01 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare papain-loaded κ-carrageenan beads for stability improvement.

Stability improvement of alpha-amylase entrapped in kappa-carrageenan beads: Physicochemical characterization and optimization using composite index

Purpose This work examines the influence of various process parameters on α-amylase entrapped in crosslinked κ-carrageenan beads for stability improvement. A three level full factorial design was employed to investigate the effect of three process variables namely κ-carrageenan concentration, potassium chloride concentration and hardening time on % entrapment, time required for 50% ( T 50) and 90% ( T 90) of enzyme release and particle size. Methods The beads were prepared by dropping the κ-carrageenan-containing α-amylase to magnetically stirred potassium chloride solution. The composite index was applied to optimize the process under study. ‘In vitro’ enzyme release profile of the beads was fitted to various release kinetics models to understand the release mechanism. Topographical characterization was carried out by SEM and entrapment was confirmed by FTIR and DSC. Stability testing according to the ICH guidelines for zone III and IV was carried out. Results With the use of ionotropic gelation method, a polymeric matrix prepared by 3.5% (w/v) κ-carrageenan, 0.7 M potassium chloride and hardening time of 30 min resulted in the production of beads characterized by disc shaped with collapsed center, absence of aggregates, % entrapment of 73.79, T 90 of 74.4 min, and composite index of 83.01. Moreover, shelf-life of the enzyme loaded beads was found to increase up to 3.53 years compared to 0.99 year of the conventional formulation. Conclusions It can be inferred that the proposed method can be used to prepare α-amylase loaded κ-carrageenan beads for stability improvement. Also the proper selection of rate-controlling carrageenan concentration and its interactive potential for crosslinking is important and will determine the overall size and shape of beads, the duration and pattern of dissolution profiles and enzyme loading capacity.

Kappa-carrageenan interactions in systems containing casein micelles and polysaccharide stabilizers

When polysaccharide food gums are added to milk-based fluid systems, phase separation of casein micelles inevitably occurs, due to biopolymer incompatibility. κ-Carrageenan can prevent bulk phase separation, although stabilized systems are still microscopically phase-separated. The mechanism by which κ-carrageenan stabilizes casein micelles from phase separation was studied in this research. Images from field emission scanning electron microscopy suggested that κ-carrageenan adhered to the casein micelle surface. Dynamic light scattering data showed casein micelle diameter increasing linearly ( r 2=0.978) as κ-carrageenan concentration increased, within the concentration range of 0–0.03% (w/w) and 3.65% casein. Phase separation experiments in 13% milk solids-not-fat (3.65% casein) −0.14% locust bean gum mixed systems with κ-carrageenan in the coil state (60 °C), κ-carrageenan in the helical state, but with helix aggregation blocked (addition of NaI), λ-carrageenan, guar (all at 0.01–0.03%) and agarose (0.01–0.05%) were used to understand the conditions in which stabilization of phase separation occurred. Our work suggests that both κ-carrageenan adsorption to casein micelle surfaces and κ-carrageenan helix aggregation are required for it to be effective at preventing casein micelle macroscopic phase separation from polysaccharides.

Phase separation in soft-serve ice cream mixes: rheology and microstructure

Abstract Soft-serve ice cream mixes containing 4% milk protein and 0.14% locust bean gum (LBG) showed extensive visual phase-separation after 21 days at 5 °C, unless κ-carrageenan was added to the formulation. Addition of κ-carrageenan (0–0.02%) did not inhibit microscopic phase separation between protein and polysaccharide. A higher degree of “emulsification” of the protein-enriched phase into the continuous serum phase was observed as the κ-carrageenan concentration increased, which correlated with inhibition of macroscopic serum separation in the mix. After macroscopic separation, the serum phase of mixes with different κ-carrageenan concentrations showed similar microstructural and rheological characteristics, implying no concentration of LBG and no presence of κ-carrageenan in the serum phase. The rheological behaviour of the protein-enriched phases indicated κ-carrageenan/casein interactions. There was no evidence of “weak-gel” formation through the addition of κ-carrageenan as a means for macroscopic stability in the system.

Interfacial compositions, microstuctures and properties of oil-in-water emulsions formed with mixtures of milk proteins and κ-carrageenan: 1. Sodium caseinate

Studies have been carried out on the changes in droplet size, surface coverage and composition, and stability of emulsions formed with 30% (w/w) soya oil, and aqueous solution containing 0.5 and 3.0% sodium caseinate and varying concentration of κ-carrageenan. In all emulsions, except those made using 3.0% sodium caseinate, addition of κ-carrageenan up to 0.4% (w/w), before homogenisation, slightly increased the average droplet size, but had no significant effect on protein surface coverage and composition. Addition of low levels of κ-carrageenan (<0.2%) caused extensive flocculation of oil droplets, which resulted in very low creaming stability. At higher κ-carrageenan concentrations, the creaming stability increased, apparently due to the formation of a network of flocculated droplets. Emulsions formed with 3% sodium caseinate without added κ-carrageenan showed extensive flocculation, due to the presence of excess unadsorbed casein molecules in the emulsion. Addition of κ-carrageenan into these emulsions resulted in gradual improvement in creaming stability. All emulsions formed without κ-carrageenan showed Newtonian behaviour (except those made with 3% sodium caseinate), as expected for unflocculated systems. The viscosity of the emulsions increased with an increase in κ-carrageenan concentration; the emulsions containing κ-carrageenan showed a clear pseudoplastic behaviour, indicating reversible flocculation of oil droplets. The creaming stability of emulsions made with mixtures of milk proteins and κ-carrageenan closely appears to be related to the structure and rheology of emulsions.

Phase Separation and Association of Globular Protein Aggregates in the Presence of Polysaccharides: 1. Mixtures of Preheated β-Lactoglobulin and κ-Carrageenan at Room Temperature

Mixtures of globular protein (β-lactoglobulin) aggregates and polysaccharides (κ-carrageenan) were studied using dynamic and static light scattering, size exclusion chromatography, and optical microscopy. Above a critical κ-carrageenan concentration the protein aggregates phase separate and form spherical microdomains, which slowly sediment. The effect of concentration and size of the protein aggregates was investigated. The phase separation is initially reversible upon dilution, but the microdomains become progressively more stable until after several days they can no longer be redissolved.

Phase behaviour of pure micellar casein/κ-carrageenan systems in milk salt ultrafiltrate

The phase behaviour of pure casein micelle/κ-carrageenan/water systems has been studied using turbidimetry, microscopy and rheology. The phase state diagram of these systems has been established at 5 and 60°C and the mechanisms are discussed. There is a significant difference between the phase diagram at 5°C and at 60°C in relation to the carrageenan concentration. At a temperature of 5°C, the data demonstrate that, depending on the relative amount of each component, the system behaves like a liquid, which can be either stable or unstable. Polymer adsorption on casein micelles at low κ-carrageenan concentration is suggested. This phenomenon is followed by depletion at higher concentrations. Interestingly, this later instability occurs at a very low concentration of κ-carrageenan (∼0.01%, w/w). As the κ-carrageenan concentration increases, the system gets trapped into a gel state. It is proposed that a casein/κ-carrageenan composite network is formed in which κ-carrageenan molecules act as bridges between casein particles. At a temperature of 60°C, when carrageenan chains adopt a coil conformation, depletion flocculation occurs when the concentration of carrageenan is around 0.3% (w/w).

Development of a fermentation process for production of a κ-carrageenase from Pseudomonas carrageenovora

The production of κ-carrageenase was studied in Pseudomonas carrageenovora strains NCIMB 302 and 9. The average specific production rate of κ-carrageenase by strain NCIMB 302 on a semidefined yeast extract medium was found to be 15 times as high as in strain 9. A linear relationship was obtained for strain NCIMB 302 in batch fermentation between the κ-carrageenase activity and the initial concentration of yeast extract. A similar linear correlation was also achieved for enzyme production on the κ-carrageenan concentration. κ-Carrageenan as an inducer could not be replaced by a low viscosity, low molecular galactose-containing carbohydrate. Supplying κ-carrageenan by fed-batch addition prolongs the time of enzyme production to a small extent, but the observed enzyme concentration was not increased. At low values, a significant increase in enzyme activity was achieved with increasing casamino acid concentration up to 3.5 g l −1, but the maximum activity decreased on further addition. When casamino acids were supplied as fed-batch, however, the production time was prolonged significantly and the enzyme activity increased 2.6 times up to 84,000 U l −1 (observed) or more than four times up to 136,000 U l −1 estimated as volume-corrected values. Substituting the casamino acids by an equal amount of a single amino acid (glutamic acid), a similar enzyme production was obtained. The highest observed enzyme activity obtained here is 17 times higher than the values reported earlier (or 27 times higher when volume-corrected values are assessed).

Survival of lac-lux marked Pseudomonas aeruginosa UG2Lr cells encapsulated in κ-carrageenan and alginate

A milder method for encapsulating viable bacterial cells in κ-carrageenan was developed. A 1% (w/v) κ-carrageenan concentration amended with clay and skim milk was used as a lower-cost alternative to alginate for potential introductions of bacteria into soil. Survival of lac-lux marked Pseudomonas aeruginosa UG2Lr cells during long-term storage in dried κ-carrageenan and alginate beads at 4°C was compared. Survival of UG2Lr cells in the κ-carrageenan formulated beads at Log 8.3 CFU/g dry beads was significantly better in the first 90 days than that in alginate at Log 7.8 CFU/g dry beads. After 360 days survival remained stable at Log 7.8 CFU/g dry beads and was not significantly different between the two carriers. Electron micrographs revealed bacterial cells in the κ-carrageenan matrix, and degradation of the bead surface after 4 weeks incubation in non-sterile soil.

Effect of galactomannan addition on the thermal behaviour of κ-carrageenan gels

Gelation/melting cycles of κ-carrageenan/galactomannan (guar, tara and locust bean gums) binary systems have been studied by measuring dynamic rheological parameters. Two experimental conditions were used, (i) the total polysaccharide concentration was kept at 1% and the κ-carrageenan/galactomannan ratio fixed at 4:1 and (ii) the κ-carrageenan concentration was fixed at 0·75% and the galactomannan content varied from 0% to 1·2%. A thermal hysteresis was observed for all mixed systems and was found to depend on the galactomannan used. From a comparison of the gelation temperature ( T g) and melting temperature ( T m) to values obtained with κ-carrageenan alone, it was suggested that galactomannan interferes with gel structure by the formation of a secondary network provided that the M/G ratio is high enough.

Modern theories of fermentation, with notes on the morphology and culture of yeasts

The production of κ-carrageenase was studied in Pseudomonas carrageenovora strains NCIMB 302 and 9. The average specific production rate of κ-carrageenase by strain NCIMB 302 on a semidefined yeast extract medium was found to be 15 times as high as in strain 9. A linear relationship was obtained for strain NCIMB 302 in batch fermentation between the κ-carrageenase activity and the initial concentration of yeast extract. A similar linear correlation was also achieved for enzyme production on the κ-carrageenan concentration. κ-Carrageenan as an inducer could not be replaced by a low viscosity, low molecular galactose-containing carbohydrate. Supplying κ-carrageenan by fed-batch addition prolongs the time of enzyme production to a small extent, but the observed enzyme concentration was not increased. At low values, a significant increase in enzyme activity was achieved with increasing casamino acid concentration up to 3.5 g l−1, but the maximum activity decreased on further addition. When casamino acids were supplied as fed-batch, however, the production time was prolonged significantly and the enzyme activity increased 2.6 times up to 84,000 U l−1 (observed) or more than four times up to 136,000 U l−1 estimated as volume-corrected values. Substituting the casamino acids by an equal amount of a single amino acid (glutamic acid), a similar enzyme production was obtained. The highest observed enzyme activity obtained here is 17 times higher than the values reported earlier (or 27 times higher when volume-corrected values are assessed).